Afferent Innervation Research Articles

Spinal Afferent Innervation From Left Dorsal Root Ganglia in the Flat‐Mounts of Whole Atria of Rats: Anterograde Tracing

ABSTRACTThe spinal afferent innervation of the heart helps to regulate cardiac functions by sending sensory information through the dorsal root ganglia (DRG) to the brain. However, the distribution and morphology of spinal afferents in the heart are not well characterized due to tracer selections, the surgical access to upper thoracic DRGs, and the thickness of the heart tissues. In this study, we injected tracer dextran biotin (DB) into the left DRGs (C8‐T3) of male Sprague–Dawley rats (3–5 months). After 16 days, flat‐mounts of the whole left and right atria were prepared and diaminobenzidine stained. Then, the DB‐labeled axons in the tissues were imaged, traced, and digitized using the Neurolucida system. Our results showed that the DB‐labeled axons from left DRGs entered the left precaval vein and projected to the left and right atria, with predominant projection in the left atrial wall. DB‐labeled varicose axons were observed in different layers, mostly in the epicardium and myocardium, but much less in the endocardium. In those layers, these spinal afferent axons branched out into simple to complex terminal arborizations, forming close appositions with cardiac muscles, intrinsic cardiac ganglia, blood vessels, and fat tissue. This work, for the first time, characterized cardiac spinal afferent distribution of the rat atria using anterograde tracing, which will provide the foundation for future studies of topographical cardiac spinal afferent innervation and remodeling in heart disease models.

Resistance to age-related hearing loss in the echolocating big brown bat (Eptesicus fuscus).

Hearing mediates many behaviours critical for survival in echolocating bats, including foraging and navigation. Although most mammals are susceptible to progressive age-related hearing loss, the evolution of biosonar, which requires the ability to hear low-intensity echoes from outgoing sonar signals, may have selected against the development of hearing deficits in bats. Many echolocating bats exhibit exceptional longevity and rely on acoustic behaviours for survival to old age; however, relatively little is known about the ageing bat auditory system. In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats (Eptesicus fuscus) and measured hearing sensitivity in young and ageing bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in bats up to 12.5 years of age, demonstrated by comparable thresholds and similar ABR and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear ageing, with similar hair cell counts, afferent and efferent innervation patterns in young and ageing bats. Here, we demonstrate that big brown bats show minimal evidence for age-related hearing loss and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.

Carvedilol through ß1-Adrenoceptor blockade ameliorates glomerulonephritis via inhibition of oxidative stress, apoptosis, autophagy, ferroptosis, endoplasmic reticulum stress and inflammation

Glomerulonephritis (GN) is one of the main causes of end stage renal disease and requires an effective treatment for inhibiting GN. Renal nerves through efferent (RENA) and afferent (RANA) innervation to glomeruli regulate the glomerular function. We delineated the role of RENA and RANA on anti-Thy1.1-induced GN. Female Wistar rats were divided into Control, Thy1.1 plus anti-Thy1.1, bilaterally renal nerve denervation (DNX) plus anti-Thy1.1, and topical capsaicin to bilateral renal nerves for selective ablation of RANA (DNAX) plus anti-Thy1.1. We examined RANA and RENA response to anti-Thy1.1 and compared the effect of DNX or DNAX on urinary oxidative stress, renal gp91, tyrosine hydroxylase (TH), calcitonin gene-related peptide (CGRP), apoptosis, autophagy, ferroptosis, antioxidant enzymes, endoplasmic reticulum (ER) stress and inflammation by western blot. Anti-Thy1.1 significantly enhanced RENA, but did not affect RANA. DNX significantly decreased TH and CGRP expression, whereas DNAX only reduced CGRP expression. Anti-Thy1.1 significantly increased glomerulosclerosis injury, urinary protein, electron paramagnetic resonance signals of alpha-(4-pyridyl-N-oxide)-N-tert-butylnitrone adducts, 8-isoprostane and nitrotyrosine levels, NADPH oxidase gp91phox (gp91), macrophage/monocyte (ED-1), GRP-78, Beclin-1/LC3-II, Bax/caspase-3/poly(ADP-ribose) polymerase expression, inflammatory cytokines levels and decreased renal Copper/Zinc superoxide dismutase, Cystine/glutamate transporter (xCT) and Glutathione peroxidase 4 (GPX4) expression vs. Control. The enhanced oxidative parameters or reduced antioxidant defense by anti-Thy1.1 were significantly attenuated by DNX but not DNAX. Additionally, oral ß1-adrenoceptor antagonist-Carvedilol at an early stage reduced anti-Thy1.1 increased proteinuria level and oxidative parameters. Our data suggest that DNX and ß1-adrenoceptor antagonist-Carvedilol efficiently attenuate oxidative stress, inflammation, ER stress, autophagy, ferroptosis and apoptosis in GN.

Sympathetic Blockade for Pain Associated With Nonaxial Bone Lesions in Patients With Cancer: An Uncontrolled Cohort.

Cancer-related bone pain remains a prevalent and frequently incapacitating ailment. Although conventional approaches effectively alleviate pain in most individuals, a subset of patients may continue to experience intractable pain. Current recommendations for treating cancer-related bone pain include oral analgesics and multimodal adjuvants, radiation therapy, and, in selected cases, intrathecal therapy. Cancer-related bone pain is mediated by a proliferation of sensory and sympathetic fibers. Thus, we believe that this pain can be successfully managed with minimally invasive sympathetic blockade (SB). In a retrospective observational cohort, we reviewed patients who underwent single-shot SB for uncontrolled cancer-related bone pain despite receiving opiate analgesics and other interventions. We documented the Edmonton Symptom Assessment Scale (ESAS) ratings, the numeric rating scale (NRS) pain scores, and the morphine equivalent daily dose (MEDD) before and after SB. The final cohort included 43 patients (median age, 58 years [range, 23-86 years]) with a history of bone pain experienced for a median of 6 months (IQR, 3-12 months). Comparing before and after the SB, patients had pain reduction -6 (IQR, -7 to -4; P<.001), reduction of ESAS scores of -17 (IQR, -23 to -3; P<.001), and reduction of MEDD -57 mg (95% CI, -79 to -34; P<.001). The treatment was well tolerated. Blockade of sympathetic afferent innervation is an effective and cost-effective modality that can be safely used to palliate intractable pain in patients with malignant bone pain.

Pharmacological regeneration of sensory hair cells restores afferent innervation and vestibular function.

The sensory cells that transduce the signals for hearing and balance are highly specialized mechanoreceptors called hair cells that together with supporting cells comprise the sensory epithelia of the inner ear. Loss of hair cells from toxin exposure and age can cause balance disorders and is essentially irreversible due to the inability of mammalian vestibular organs to regenerate physiologically active hair cells. Here, we show substantial regeneration of hair cells in a mouse model of vestibular damage by treatment with a combination of glycogen synthase kinase 3β and histone deacetylase inhibitors. The drugs stimulated supporting cell proliferation and differentiation into hair cells. The new hair cells were reinnervated by vestibular afferent neurons, rescuing otolith function by restoring head translation-evoked otolith afferent responses and vestibuloocular reflexes. Drugs that regenerate hair cells thus represent a potential therapeutic approach to the treatment of balance disorders.

Resistance to age-related hearing loss in the echolocating big brown bat ( Eptesicus fuscus ).

Hearing mediates many behaviors critical for survival in echolocating bats, including foraging and navigation. Most mammals are susceptible to progressive age-related hearing loss; however, the evolution of biosonar, which requires the ability to hear low-intensity echoes from outgoing sonar signals, may have selected against the development of hearing deficits in echolocating bats. Although many echolocating bats exhibit exceptional longevity and rely on acoustic behaviors for survival to old age, relatively little is known about the aging bat auditory system. In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats ( Eptesicus fuscus ) and measured hearing sensitivity in young and aging bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in aging bats, demonstrated by comparable thresholds and similar ABR wave and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear aging, with similar hair cell counts, afferent, and efferent innervation patterns in young and aging bats. Here we demonstrate that big brown bats show minimal evidence for age-related loss of peripheral hearing sensitivity and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.

(037) VESTIBULODYNIA SECONDARY TO LUMBOSACRAL ANNULAR TEAR-INDUCED SACRAL RADICULOPATHY

Abstract Introduction Vestibulodynia is a prevalent problem in individuals with vulvas, with as many as 30% reporting complaints consistent with entry dyspareunia. Distinctive vestibulodynia pathophysiologies may include psychosocial factors, pelvic floor muscle dysfunction, dermatologic disorders, inflammatory disorders, hormonal deficiencies, neuroproliferative pathology, and pudendal neuropathy. Sacral radiculopathy is considered an inflammatory irritation of S2-S3 nerve roots in the cauda equina by various pathologies such as lumbosacral annular tear. S2-S3 nerve roots are formed by the convergence of the pelvic, pudendal, and sciatic nerves and genito-pelvic clinical symptoms may result from inflammatory involvement of the sensory fields of these nerves. Vestibulodynia may occur from sacral radiculopathy since the somatic and visceral afferent innervation of the vestibule occurs via the pudendal and pelvic nerves, respectively; however, to the best of our knowledge, there has not been a case series of such patients previously reported. Objective To perform a chart review of patients with vestibulodynia secondary to lumbosacral annular tear-induced sacral radiculopathy. Methods Vestibulodynia patients who presented to our sexual medicine clinic were evaluated by detailed history, vulvoscopy, vestibular anesthesia testing, neurogenital testing and lumbosacral MRI. When these tests warranted it, a transforaminal epidural spinal injection with 1 ml lidocaine 1% was performed to substantiate the diagnosis. If appropriate, spine surgery was performed. Improvement was assessed using the Patient Global Impression of Improvement (PGI-I). Results Four patients, 3 cis-gender women and 1 trans-man, mean age 33 years (range 25-42), were diagnosed with vestibulodynia secondary to lumbosacral annular tear-induced sacral radiculopathy. Concomitant sacral radiculopathy symptoms included: clitoral hypersensitivity (50%), bladder urgency/frequency (75%), umbilical hypersensitivity (75%), vaginal dysesthesia (25%), vulvar itching (50%), low back pain (100%), and sciatica (75%). Vulvoscopy was performed to exclude other forms of vestibulodynia. All patients had: a) positive cotton-tipped swab testing throughout the entire vestibule; b) negative vestibular anesthesia testing; c) abnormal neurogenital testing; d) lumbosacral MRI that revealed surgically treatable lumbosacral annular tear (L5-S1, L4-L5); e) transforaminal epidural spinal injection resulting in clinically significant symptom reduction. Lumbar endoscopic spine surgery resulted in improvement in 75% reporting PGI-I 1-3 (very much better - somewhat better). Conclusions We report the first series of patients with vestibulodynia resulting from lumbosacral annular tear-induced sacral radiculopathy. Clinicians should be aware that bothersome vestibulodynia in patients with vulvas can occur from cauda equina pathology (Region 3) outside the genitals (Region 1) and/or pelvis/perineum (Region 2). Disclosure Any of the authors act as a consultant, employee or shareholder of an industry for: Elliquence.

Identification of Spinal Afferent Innervation in the Rat Heart: Atria and Ventricles: Anterograde Tracing

The spinal afferent innervation of the heart regulates cardiac functions by sending sensory information from the dorsal root ganglia (DRG) to the brain. However, the distribution and morphology of spinal afferents in the whole heart are not well characterized. The challenges here are: 1) diffculty in surgically accessing the upper thoracic DRG to label only cardiac spinal afferents. 2) handling flat-mounts of whole atria and ventricles (thickness ~850 μm) without using sections or partial tissues that could disrupt the continuity of axonal structures. To overcome these challenges, we injected anterograde tracer dextran biotin (DB) into the left DRG (C8-T3) of male SD rats (3-5 months) and allowed them to recover for 14 days for tracer transportation. Flat-mounts of the left and right atria and ventricles were prepared, and DAB (3,3′-Diaminobenzidine) stained, followed by imaging, tracing, and digitization using the Neurolucida system. Our findings are: In the atria, the DB-labeled axons entered the atria potentially through the left precaval vein (LPCV), with predominant innervation of the left side of the atrial wall and extended their projection towards the auricles, middle region of the atrium, and pulmonary veins. Most of the spinal afferent axons were present in the myocardial layer. Varicose DB-labeled axons were also observed on cardiac targets at different regions, and distinct morphological structures were found. In the cardiac muscle, DB-labeled axons ran mainly in the direction of muscle fibers. Also, varicose axons ran along the small vasculature on the atria. Spinal afferents formed varicose contacts with individual principal neurons within some intrinsic cardiac ganglia (ICG). In the ventricles, DB-labeled axons were present in the left and right ventricles, which ramified and expressed spherical-like varicosities on the cardiac muscle. In both the atria and ventricles, spinal afferent axons formed simple, branching, and complex terminal structures. Thus, our work, for the first time, successfully labeled, traced, and digitized the spinal cardiac afferents in the flat-mounts of whole atria and ventricles, which will provide a foundation for specific labeling of cardiac spinal afferent fibers. In addition, it will lead to the future study of topographical cardiac spinal afferent innervation, functional studies, and their remodeling in pathological conditions. This study was supported by NIH HEAL/SPARC U01 NS113867-01, NIH R15 R15HL137143-01A1, and UCF dissertation completion Fellowships. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Perineuronal net in the extrinsic innervation of the distal colon of mice and its remodeling in ulcerative colitis.

The perineuronal net (PNN) is a well-described highly specialized extracellular matrix structure found in the central nervous system. Thus far, no reports of its presence or connection to pathological processes have been described in the peripheral nervous system. Our study demonstrates the presence of a PNN in the spinal afferent innervation of the distal colon of mice and characterizes structural and morphological alterations induced in an ulcerative colitis (UC) model. C57Bl/6 mice were given 3% dextran sulfate sodium (DSS) to induce acute or chronic UC. L6/S1 dorsal root ganglia (DRG) were collected. PNNs were labeled using fluorescein-conjugated Wisteria Floribunda (WFA) l lectin, and calcitonin gene-related peptide (CGRP) immunofluorescence was used to detect DRG neurons. Most DRG cell bodies and their extensions toward peripheral nerves were found surrounded by the PNN-like structure (WFA+), labeling neurons' cytoplasm and the pericellular surfaces. The amount of WFA+ neuronal cell bodies was increased in both acute and chronic UC, and the PNN-like structure around cell bodies was thicker in UC groups. In conclusion, a PNN-like structure around DRG neuronal cell bodies was described and found modulated by UC, as changes in quantity, morphology, and expression profile of the PNN were detected, suggesting a potential role in sensory neuron peripheral sensitization, possibly modulating the pain profile of ulcerative colitis.

Expression and function of transient receptor potential melastatin 3 in the spinal afferent innervation of the mouse colon.

Abdominal pain is a cardinal symptom of inflammatory bowel disease (IBD). Transient receptor potential (TRP) channels contribute to abdominal pain in preclinical models of IBD, and TRP melastatin 3 (TRPM3) has recently been implicated in inflammatory bladder and joint pain in rodents. We hypothesized that TRPM3 is involved in colonic sensation and is sensitized during colitis. We used immunohistochemistry, ratiometric Ca2+ imaging, and colonic afferent nerve recordings in mice to evaluate TRPM3 protein expression in colon-projecting dorsal root ganglion (DRG) neurons, as well as functional activity in DRG neurons and colonic afferent nerves. Colitis was induced using dextran sulfate sodium (DSS) in drinking water. TRPM3 protein expression was observed in 76% of colon-projecting DRG neurons and was often colocalized with calcitonin gene-related peptide. The magnitudes of intracellular Ca2+ transients in DRG neurons in response to the TRPM3 agonists CIM-0216 and pregnenolone sulfate sodium were significantly greater in neurons from mice with colitis compared with controls. In addition, the percentage of DRG neurons from mice with colitis that responded to CIM-0216 was significantly increased. CIM-0216 also increased the firing rate of colonic afferent nerves from control and mice with colitis. The TRPM3 inhibitor isosakuranetin inhibited the mechanosensitive response to distension of wide dynamic range afferent nerve units from mice with colitis but had no effect in control mice. Thus, TRPM3 contributes to colonic sensory transduction and may be a potential target for treating pain in IBD.NEW & NOTEWORTHY This is the first study to characterize TRPM3 protein expression and function in colon-projecting DRG neurons. A TRPM3 agonist excited DRG neurons and colonic afferent nerves from healthy mice. TRPM3 agonist responses in DRG neurons were elevated during colitis. Inhibiting TRPM3 reduced the firing of wide dynamic range afferent nerves from mice with colitis but had no effect in control mice.

Human liver afferent and efferent nerves revealed by 3-D/Airyscan super-resolution imaging.

Neural regulation of hepatic metabolism has long been recognized. However, the detailed afferent and efferent innervation of the human liver has not been systematically characterized. This is largely due to the liver's high lipid and pigment contents, causing false-negative (light scattering and absorption) and false-positive (autofluorescence) results in in-depth fluorescence imaging. Here, to avoid the artifacts in three-dimensional (3-D) liver neurohistology, we embed the bleached human liver in the high-refractive-index polymer for tissue clearing and antifade 3-D/Airyscan super-resolution imaging. Importantly, using the paired substance P (SP, sensory marker) and PGP9.5 (pan-neuronal marker) labeling, we detect the sensory nerves in the portal space, featuring the SP+ varicosities in the PGP9.5+ nerve bundles/fibers, confirming the afferent liver innervation. Also, using the tyrosine hydroxylase (TH, sympathetic marker) labeling, we identify 1) condensed TH+ sympathetic nerves in the portal space, 2) extension of sympathetic nerves from the portal to the intralobular space, in which the TH+ nerve density is 2.6 ± 0.7-fold higher than that of the intralobular space in the human pancreas, and 3) the TH+ nerve fibers and varicosities contacting the ballooning cells, implicating potential sympathetic influence on hepatocytes with macrovesicular fatty change. Finally, using the vesicular acetylcholine transporter (VAChT, parasympathetic marker), PGP9.5, and CK19 (epithelial marker) labeling with panoramic-to-Airyscan super-resolution imaging, we detect and confirm the parasympathetic innervation of the septal bile duct. Overall, our labeling and 3-D/Airyscan imaging approach reveal the hepatic sensory (afferent) and sympathetic and parasympathetic (efferent) innervation, establishing a clinically related setting for high-resolution 3-D liver neurohistology.NEW & NOTEWORTHY We embed the human liver (vs. pancreas, positive control) in the high-refractive-index polymer for tissue clearing and antifade 3-D/Airyscan super-resolution neurohistology. The pancreas-liver comparison reveals: 1) sensory nerves in the hepatoportal space; 2) intralobular sympathetic innervation, including the nerve fibers and varicosities contacting the ballooning hepatocytes; and 3) parasympathetic innervation of the septal bile duct. Our results highlight the sensitivity and resolving power of 3-D/Airyscan super-resolution imaging in human liver neurohistology.

Sensory innervation of the human shoulder joints in healthy and in chronic pain shoulder syndromes

BackgroundAfferent innervation of shoulder joints plays a fundamental role in nociception and mechanoception and its alteration result in shoulder´s disease that course with pain and functional disability. MethodsJoints shoulder from healthy subjects (n = 20) and with chronic pain shoulder syndromes (n = 17) were analyzed using immunohistochemistry for S100 protein to identify nerve structures (nerve fibers and sensory corpuscles), coupled with a quantification of the sensory formations. Sensory nerve formations were quantified in 13 distinct areas in healthy joint shoulder and in the available equivalent areas in the pathological joints. Statistical analyses were conducted to assess differences between healthy shoulder and pathological shoulder joint (p< 0.05). ResultsAll analyzed structures, i.e., glenohumeral capsule, acromioclavicular capsule, the extraarticular structures (subcoracoid region and subacromio-subdeltoid bursa) and intraarticular structures (biceps brachii tendon and labrum articulare) are variably innervated except the extrinsic coracoacromial ligament, which was aneural. The afferent innervation of healthy human shoulder joints consists of free nerve endings, simple lamellar corpuscles and Ruffini’s corpuscles. Occasionally, Golgi-Mazzoni’s and Pacinian corpuscles were found. However, the relative density of each one varied among joints and/or the different zones within the same joint. As a rule, the upper half and anterior half of healthy glenohumeral capsules have a higher innervation compared to the lower and posterior respectably. On the other hand, in joints from subjects suffering chronic shoulder pain, a reduced innervation was found, involving more the corpuscles than free nerve endings. ConclusionsOur findings report a global innervation map of the human shoulder joints, especially the glenohumeral one, and this knowledge might be of interest for arthroscopic surgeons allowing to develop more selective and unhurt treatments, controlling the pain, and avoiding the loss of afferent innervation after surgical procedures. To the light of our results the postero-inferior glenohumeral capsular region seems to be the more adequate to be a surgical portal (surgical access area) to prevent nerve lesions.

Sprouting of afferent and efferent inputs to pelvic organs after spinal cord injury.

Neural plasticity occurs within the central and peripheral nervous systems after spinal cord injury (SCI). Although central alterations have extensively been studied, it is largely unknown whether afferent and efferent fibers in pelvic viscera undergo similar morphological changes. Using a rat spinal cord transection model, we conducted immunohistochemistry to investigate afferent and efferent innervations to the kidney, colon, and bladder. Approximately 3-4 weeks after injury, immunostaining demonstrated that tyrosine hydroxylase (TH)-labeled postganglionic sympathetic fibers and calcitonin gene-related peptide (CGRP)-immunoreactive sensory terminals sprout in the renal pelvis and colon. Morphologically, sprouted afferent or efferent projections showed a disorganized structure. In the bladder, however, denser CGRP-positive primary sensory fibers emerged in rats with SCI, whereas TH-positive sympathetic efferent fibers did not change. Numerous CGRP-positive afferents were observed in the muscle layer and the lamina propria of the bladder following SCI. TH-positive efferent inputs displayed hypertrophy with large diameters, but their innervation patterns were sustained. Collectively, afferent or efferent inputs sprout widely in the pelvic organs after SCI, which may be one of the morphological bases underlying functional adaptation or maladaptation.

Sex differences in zymosan-induced behavioral visceral hypersensitivity and colorectal afferent sensitization.

Sex differences in visceral nociception have been reported in clinical and preclinical studies, but the potential differences in sensory neural encoding of the colorectum between males and females are not well understood. In this study, we systematically assessed sex differences in colorectal neural encoding by conducting high-throughput optical recordings in intact dorsal root ganglia (DRGs) from control and visceral hypersensitive mice. We found an apparent sex difference in zymosan-induced behavioral visceral hypersensitivity: enhanced visceromotor responses to colorectal distension were observed only in male mice, not in female mice. In addition, a higher number of mechanosensitive colorectal afferents were identified per mouse in the zymosan-treated male group than in the saline-treated male group, whereas the mechanosensitive afferents identified per mouse were comparable between the zymosan- and saline-treated female groups. The increased number of identified afferents in zymosan-treated male mice was predominantly from thoracolumbar (TL) innervation, which agrees with the significant increase in the TL afferent proportion in the zymosan group as compared with the control group in male mice. In contrast, female mice showed no difference in the proportion of colorectal neurons between saline- and zymosan-treated groups. Our results revealed a significant sex difference in colorectal afferent innervation and sensitization in the context of behavioral visceral hypersensitivity, which could drive differential clinical symptoms in male and female patients.NEW & NOTEWORTHY We used high-throughput GCaMP6f recordings to study 2,275 mechanosensitive colorectal afferents in mice. Our results revealed significant sex differences in the zymosan-induced behavioral visceral hypersensitivity, which were present in male but not female mice. Male mice also showed sensitization of colorectal afferents in the thoracolumbar pathway, whereas female mice did not. These findings highlight sex differences in sensory neural anatomy and function of the colorectum, with implications for sex-specific therapies for treating visceral pain.

Spinal afferent innervation in flat-mounts of the rat stomach: anterograde tracing

The dorsal root ganglia (DRG) project spinal afferent axons to the stomach. However, the distribution and morphology of spinal afferent axons in the stomach have not been well characterized. In this study, we used a combination of state-of-the-art techniques, including anterograde tracer injection into the left DRG T7-T11, avidin–biotin and Cuprolinic Blue labeling, Zeiss M2 Imager, and Neurolucida to characterize spinal afferent axons in flat-mounts of the whole rat stomach muscular wall. We found that spinal afferent axons innervated all regions with a variety of distinct terminal structures innervating different gastric targets: (1) The ganglionic type: some axons formed varicose contacts with individual neurons within myenteric ganglia. (2) The muscle type: most axons ran in parallel with the longitudinal and circular muscles and expressed spherical varicosities. Complex terminal structures were observed within the circular muscle layer. (3) The ganglia-muscle mixed type: some individual varicose axons innervated both myenteric neurons and the circular muscle, exhibiting polymorphic terminal structures. (4) The vascular type: individual varicose axons ran along the blood vessels and occasionally traversed the vessel wall. This work provides a foundation for future topographical anatomical and functional mapping of spinal afferent axon innervation of the stomach under normal and pathophysiological conditions.

Vagal afferent responses to cystometry in rat models of urinary bladder irritation: c-fos immunohistochemistry study.

Although recent literature provides increasing evidence concerning urinary bladder innervation by vagal afferents, the functional aspects and the conditions at which these afferents are recruited are still unclear. In the present study, the neuronal responses of nodose ganglion following cystometry, under different models of rat's urinary bladder irritation, cyclophosphamide (CYP), cyclophosphamide with cervical vagotomy (Vx), chronic HCl, and acute HCl, were investigated using c-fos immunohistochemistry. The c-fos expression in the nodose ganglion, following cystometry, was increased significantly in the CYP and chronic-HCl groups compared to the intact, Vx, and acute-HCl groups. In addition, the acute-HCl group showed a significant increase compared to intact animals. Following cervical vagotomy, the expression in the Vx group decreased significantly compared to the CYP group, but was significantly higher than that in the intact group. The results of this study demonstrate the innervation of the vagus afferents to the urinary bladder. This innervation is activated under urinary bladder irritation conditions, which may indicate a possible role of the vagus nerve during urinary bladder pathology.

Oncomodulin regulates spontaneous calcium signalling and maturation of afferent innervation in cochlear outer hair cells.

Cochlear outer hair cells (OHCs) are responsible for the exquisite frequency selectivity and sensitivity of mammalian hearing. During development, the maturation of OHC afferent connectivity is refined by coordinated spontaneous Ca2+ activity in both sensory and non-sensory cells. Calcium signalling in neonatal OHCs can be modulated by oncomodulin (OCM, β-parvalbumin), an EF-hand calcium-binding protein. Here, we investigated whether OCM regulates OHC spontaneous Ca2+ activity and afferent connectivity during development. Using a genetically encoded Ca2+ sensor (GCaMP6s) expressed in OHCs in wild-type (Ocm+/+ ) and Ocm knockout (Ocm-/- ) littermates, we found increased spontaneous Ca2+ activity and upregulation of purinergic receptors in OHCs from Ocm-/- cochlea immediately following birth. The afferent synaptic maturation of OHCs was delayed in the absence of OCM, leading to an increased number of ribbon synapses and afferent fibres on Ocm-/- OHCs before hearing onset. We propose that OCM regulates the spontaneous Ca2+ signalling in the developing cochlea and the maturation of OHC afferent innervation. KEY POINTS: Cochlear outer hair cells (OHCs) exhibit spontaneous Ca2+ activity during a narrow period of neonatal development. OHC afferent maturation and connectivity requires spontaneous Ca2+ activity. Oncomodulin (OCM, β-parvalbumin), an EF-hand calcium-binding protein, modulates Ca2+ signals in immature OHCs. Using transgenic mice that endogenously expressed a Ca2+ sensor, GCaMP6s, we found increased spontaneous Ca2+ activity and upregulated purinergic receptors in Ocm-/- OHCs. The maturation of afferent synapses in Ocm-/- OHCs was also delayed, leading to an upregulation of ribbon synapses and afferent fibres in Ocm-/- OHCs before hearing onset. We propose that OCM plays an important role in modulating Ca2+ activity, expression of Ca2+ channels and afferent innervation in developing OHCs.

The role of sympathetic innervation in neonatal muscle growth and neuromuscular contractures.

Neonatal brachial plexus injury (NBPI), a leading cause of pediatric upper limb paralysis, results in disabling and incurable muscle contractures that are driven by impaired longitudinal growth of denervated muscles. A rare form of NBPI, which maintains both afferent and sympathetic muscle innervation despite motor denervation, protects against contractures. We have previously ruled out a role for NRG/ErbB signaling, the predominant pathway governing antegrade afferent neuromuscular transmission, in modulating the formation of contractures. Our current study therefore investigated the contributions of sympathetic innervation of skeletal muscle in modulating NBPI-induced contractures. Through chemical sympathectomy and pharmacologic modification with a β2 -adrenergic agonist, we discovered that sympathetic innervation alone is neither required nor sufficient to modulate contracture formation in neonatal mice. Despite this, sympathetic innervation plays an intriguing sex-specific role in mediating neonatal muscle growth, as the cross-sectional area (CSA) and volume of normally innervated male muscles were diminished by ablation of sympathetic neurons and increased by β-adrenergic stimulation. Intriguingly, the robust alterations in CSA occurred with minimal changes to normal longitudinal muscle growth as determined by sarcomere length. Instead, β-adrenergic stimulation exacerbated sarcomere overstretch in denervated male muscles, indicating potentially discrete regulation of muscle width and length. Future investigations into the mechanistic underpinnings of these distinct aspects of muscle growth are thus essential for improving clinical outcomes in patients affected by muscle disorders in which both length and width are affected.

Regulation of cerebellar network development by granule cells and their molecules.

The well-organized cerebellar structures and neuronal networks are likely crucial for their functions in motor coordination, motor learning, cognition, and emotion. Such cerebellar structures and neuronal networks are formed during developmental periods through orchestrated mechanisms, which include not only cell-autonomous programs but also interactions between the same or different types of neurons. Cerebellar granule cells (GCs) are the most numerous neurons in the brain and are generated through intensive cell division of GC precursors (GCPs) during postnatal developmental periods. While GCs go through their own developmental processes of proliferation, differentiation, migration, and maturation, they also play a crucial role in cerebellar development. One of the best-characterized contributions is the enlargement and foliation of the cerebellum through massive proliferation of GCPs. In addition to this contribution, studies have shown that immature GCs and GCPs regulate multiple factors in the developing cerebellum, such as the development of other types of cerebellar neurons or the establishment of afferent innervations. These studies have often found impairments of cerebellar development in animals lacking expression of certain molecules in GCs, suggesting that the regulations are mediated by molecules that are secreted from or present in GCs. Given the growing recognition of GCs as regulators of cerebellar development, this review will summarize our current understanding of cerebellar development regulated by GCs and molecules in GCs, based on accumulated studies and recent findings, and will discuss their potential further contributions.

High-resolution volumetric imaging constrains compartmental models to explore synaptic integration and temporal processing by cochlear nucleus globular bushy cells.

Globular bushy cells (GBCs) of the cochlear nucleus play central roles in the temporal processing of sound. Despite investigation over many decades, fundamental questions remain about their dendrite structure, afferent innervation, and integration of synaptic inputs. Here, we use volume electron microscopy (EM) of the mouse cochlear nucleus to construct synaptic maps that precisely specify convergence ratios and synaptic weights for auditory nerve innervation and accurate surface areas of all postsynaptic compartments. Detailed biophysically based compartmental models can help develop hypotheses regarding how GBCs integrate inputs to yield their recorded responses to sound. We established a pipeline to export a precise reconstruction of auditory nerve axons and their endbulb terminals together with high-resolution dendrite, soma, and axon reconstructions into biophysically detailed compartmental models that could be activated by a standard cochlear transduction model. With these constraints, the models predict auditory nerve input profiles whereby all endbulbs onto a GBC are subthreshold (coincidence detection mode), or one or two inputs are suprathreshold (mixed mode). The models also predict the relative importance of dendrite geometry, soma size, and axon initial segment length in setting action potential threshold and generating heterogeneity in sound-evoked responses, and thereby propose mechanisms by which GBCs may homeostatically adjust their excitability. Volume EM also reveals new dendritic structures and dendrites that lack innervation. This framework defines a pathway from subcellular morphology to synaptic connectivity, and facilitates investigation into the roles of specific cellular features in sound encoding. We also clarify the need for new experimental measurements to provide missing cellular parameters, and predict responses to sound for further in vivo studies, thereby serving as a template for investigation of other neuron classes.